Histone deacetylases (HDACs) deacetylate histones and non-histone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6-deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild-type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of α-tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin-remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting.